CN105950610A - Method for rapidly extracting fecal bacterial genome DNA and virus RNA through paramagnetic particle method - Google Patents
Method for rapidly extracting fecal bacterial genome DNA and virus RNA through paramagnetic particle method Download PDFInfo
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- CN105950610A CN105950610A CN201610402154.2A CN201610402154A CN105950610A CN 105950610 A CN105950610 A CN 105950610A CN 201610402154 A CN201610402154 A CN 201610402154A CN 105950610 A CN105950610 A CN 105950610A
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- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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Abstract
The invention discloses a method for rapidly extracting fecal bacterial genome DNA and virus RNA through a paramagnetic particle method. The method comprises the steps that 1, faeces is taken and put into a centrifuge tube, a buffer A is added, crushing and standing are conducted, and supernatant is taken and put into a new centrifuge tube; 2, a buffer B and a buffer C are added into the supernatant in sequence, and water-bath heating is conducted; 3, adsorption of DAN and RNA is conducted, wherein after decomposition is completed, a buffer D and a buffer E are added in sequence, vortex oscillation, standing and magnetic separation are conducted, and solid substances are left; 4, a buffer F and a buffer G are added into a centrifuge tube with left solid substances in sequence, vortex oscillation and magnetic separation are conducted; 5, a buffer H is added into the centrifuge tube subjected to treatment in the fourth step, vortex oscillation and water-bath heating are conducted, vortex oscillation is conducted again, cooling and magnetic separation are conducted, and the supernatant is moved to a new centrifuge tube for use.
Description
Technical field
The present invention relates to molecular biology, medicine technology field, relate in particular to a kind of paramagnetic particle method fecal bacteria genomic DNA/
Viral RNA rapid extracting method.
Background technology
Intestinal microbial population is the important component part of body, and the change of its Bacterial community value volume and range of product has with the health of body and disease
Substantial connection, the health status of humans and animals can be the most mutual by the external environment factor and body self and internal microorganism flora
Effect determines, therefore the interaction relationship between research host and internal flora is significant.
Feces is similar to intestinal contents, containing substantial amounts of microorganism, plant and Colonic exfoliative cells etc., is made by fecal microorganism
For object of study, be conducive to preferably studying enteric microorganism, and stool sampling is convenient, not damaged.
Due to feces complicated component, constituent and the structure of different bacteria wall are different, so extracting method is to obtaining gene
Group DNA has the biggest difference.And most critical most important one during the extraction of DNA or RNA is enteric microorganism follow-up study in sample
Step, not only wants microbial DNA as much as possible or RNA to extract, more to ensure the quality extracted, remove other during extraction
The a large amount of degradeds during extracting are avoided in the extraction of impurity, especially RNA, the most quickly, conveniently, efficient feces gene
The extracting method of group DNA/RNA becomes extremely important.
The method extracting faeces DNA/RNA at present is broadly divided into traditional method and various RNA isolation kit, but traditional method step is numerous
Trivial, waste time and energy, RNA isolation kit expensive and extract faeces DNA/RNA yield is the highest, purity is low, time length, effect
The most undesirable.
Summary of the invention
Present invention aims to the defect of above-mentioned prior art, it is provided that a kind of paramagnetic particle method fecal bacteria genomic DNA/virus
RNA rapid extracting method, overcomes traditional method complex steps in prior art, wastes time and energy, and RNA isolation kit is expensive and carries
The defect that faeces DNA/RNA yield is the highest, purity is low, time length, effect are undesirable taken out.
To achieve these goals, the technical scheme is that
A kind of paramagnetic particle method fecal bacteria genomic DNA/viral RNA rapid extracting method, the step of this extracting method is:
S1, feces pre-treatment: weigh human or animal's feces in centrifuge tube, add buffer A, treat that feces fully crushes laggard
Row stands, until centrifuge tube solid precipitation, have supernatant to separate out time, take supernatant in new centrifuge tube, in case subsequent experimental；
Described buffer A by concentration be 0.1～1M potassium dihydrogen phosphate and sodium chloride solution 1:1 by volume that concentration is 0.1～1M～
5 mix；
The cracking of microorganism in S2, feces: successively add buffer B and buffer C in the centrifuge tube holding above-mentioned supernatant,
Vortex oscillation mix homogeneously, heating in water bath under conditions of less than 100° centigrade；Described buffer B is by concentration 0.1～10mol/L
Guanidinesalt, concentration 1～50mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride and the sodium lauryl sulphate of concentration 10～100mmol/L
1:1～3:1～5 mixes by volume；PH value range is 4.0～7.0；Described buffer C is 1～50mg/mL egg
White enzyme K；
The absorption of S3, DNA/RNA: after having cracked, successively adds buffer D and buffer E, and vortex oscillation, fully
Stand after mixing；Then the centrifuge tube after standing is put into and is carried out Magneto separate on magnetic frame, removes the liquid in centrifuge tube, stays
Solids；Described buffer D is magnetic bead；Described buffer E is volumetric concentration 20～the alcoholic solution of 80%；
S4, the removal of residual impurity: successively adding buffer F and buffer G to the centrifuge tube leaving solids, vortex shakes
Swing, fully carry out Magneto separate after mixing；Described buffer F is by concentration 0.1～10mol/L saline solution and concentration 1～50mmol/L
Tri(Hydroxymethyl) Amino Methane Hydrochloride 1:1 by volume～5 mixes, and pH value is 5～10；Described Buffer G be 1～
The Tri(Hydroxymethyl) Amino Methane Hydrochloride solution of 50mmol/L；
The eluting of S5, DNA/RNA: add buffer H, vortex oscillation in the centrifuge tube after processing through step 4, wait to fill
Heating in water bath is carried out after dividing mixing；Then it is vortexed again for vibration, fully mixes, carry out Magneto separate after being cooled to room temperature, contained
There is the eluent of genome NDA/RNA, eluent is moved on in new centrifuge tube standby；Described buffer H be concentration 0.1～
10mol/L HCI solution, pH value is 6～10.
As the improvement to technique scheme, in step 4, when the pH value range of buffer F is not between 5～10,
The pH value range of the alcoholic solution regulation buffer F adding 30%～80% is 5～10.
As the improvement to technique scheme, in step 2, the temperature of heating in water bath is 37 DEG C～95 DEG C；Heating in water bath
Time is 10 minutes to 1 hour.
As the improvement to technique scheme, in steps of 5, the temperature of heating in water bath is 30 DEG C～75 DEG C；Heating in water bath
Time is 1 minute to 10 minutes.
As the improvement to technique scheme, in step 2, in 3,4,5, the time of vortex oscillation is 3～30 seconds.
As the improvement to technique scheme, in step 3, the time of standing is 1～10min.
Compared with prior art, the present invention has the advantage that with good effect and is:
The paramagnetic particle method fecal bacteria genomic DNA/viral RNA rapid extracting method of the present invention, extracts bacterial genomes from feces
DNA or viral RNA speed are fast, and extracted amount is big, purity good, easy and simple to handle, price is low, steady quality, safety and environmental protection, nothing
Need to be centrifuged repeatedly, self-reacting device can be coordinated to realize high-volume automation mechanized operation.When this method solving faeces DNA/RNA extraction
Between long, complex steps, destructive big, the shortcoming such as extracted amount is low, purity difference, price are high with not enough.
Detailed description of the invention
Below in conjunction with specific embodiment of the present invention, technical scheme is clearly and completely described, it is clear that
Described embodiment is only a part of embodiment of the present invention rather than whole embodiments.Based on the embodiment in the present invention,
The every other embodiment that those of ordinary skill in the art are obtained under not making creative work premise, that is made any repaiies
Change, equivalent, improvement etc., should be included within the scope of the present invention.
Embodiment 1
The step of the extracting method of the fecal bacteria extracting genome DNA of the present embodiment is:
1. feces pre-treatment: weigh human or animal feces 200mg～500mg in 2mL centrifuge tube, adds 300ul～3mL
Buffer A, fully broken after, take supernatant 100ul～1mL in new centrifuge tube, in case subsequent experimental.
2. the cracking of microorganism in feces: add 200ul～2mL buffer B and 5～50ul in above-mentioned standby centrifuge tube
Buffer C, vortex oscillation mix homogeneously, heating in water bath 10 minutes to 1 hour under the conditions of 37 DEG C～95 DEG C.
3. Impurity removal and the absorption of DNA/RNA: add 10～100ul buffer Ds' and 100ul～1mL after having cracked
Buffer E, vortex oscillation fully mixes, and stands 1～10min.Centrifuge tube is put into Magneto separate on magnetic frame, removes centrifuge tube
In liquid.
4. the removal of residual impurity: be separately added into 100ul～1000ul buffer F and buffer G, vortex oscillation 3～30
Second, fully carry out Magneto separate after mixing.
The eluting of 5.DNA/RNA: add 30～300ul buffer H, vortex oscillation 3～30 seconds in above-mentioned centrifuge tube, fill
Divide mixing, 30～75 DEG C of water-baths 1～10 minutes.Vortex oscillation 3～30 seconds, fully mix, and is cooled to room temperature, Magneto separate,
Eluent is moved on in new centrifuge tube standby.
Buffer A: by concentration be 0.1～1M potassium dihydrogen phosphate and 0.1～1M sodium chloride solution 1:1 by volume～5 mix
Conjunction forms.
Buffer B: be by concentration 0.1～10mol/L guanidinesalt, 1～50mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride and concentration
The sodium lauryl sulphate of 10～100mmol/L 1:1～3:1 by volume～5 mixes, and pH value range is 4.0～7.0.
Buffer C:1～50mg/mL E.C. 3.4.21.64.
Buffer D: magnetic bead: magnetic is strong, can thoroughly adsorb for 1～5 second, good dispersion, vibration can realize single dispersing gently.
Buffer E: volumetric concentration is 20～80% alcoholic solution.
Buffer F: pressed body by concentration 0.1～10mol/L saline solution and 1～50mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride
Amassing and mix than 1:1～5, adjusting pH value range is 5～10, adds the alcoholic solution of 30%～80% during use.
Buffer G:1～50mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride, during use, volumetric concentration is 20～80% alcoholic solution.
Buffer H: concentration 0.1～10mol/L HCI solution, pH value is 6～10.
Embodiment 2
The step of the extracting method of the fecal bacteria extracting genome DNA of the present embodiment is:
1. feces pre-treatment: weigh human or animal feces 200mg in 2mL centrifuge tube, adds 500ul～1mL buffer A,
After fully broken, take supernatant 200ul～500ul in new centrifuge tube, in case subsequent experimental.
2. the cracking of microorganism in feces: add 200～500ul buffer B and 20ul buffer in above-mentioned standby centrifuge tube
C, vortex oscillation mixes.
Uniformly, heating in water bath 30 minutes to 1 hour under the conditions of 37 DEG C～95 DEG C.Take after having cracked supernatant 500ul～1mL in
In new centrifuge tube.
The absorption of 3.DNA: add the buffer E of 20～50ul buffer D and 300～500ul in above-mentioned centrifuge tube,
Vortex oscillation fully mixes, and stands 5min.Centrifuge tube is put into Magneto separate on magnetic frame, removes the liquid in centrifuge tube.
4. the removal of residual impurity: be sequentially added into 100ul～1000ul buffer F and buffer G, vortex oscillation 3～
30 seconds, fully carry out Magneto separate after mixing, inhale and abandon waste liquid.
The eluting of 5.DNA: addition 30～300ul buffer H in above-mentioned centrifuge tube, vortex oscillation 3～30 seconds, the most mixed
Even, 30～75 DEG C of water-baths 1～10 minutes.Vortex oscillation 3～30 seconds, fully mix, be cooled to room temperature, Magneto separate, will wash
De-liquid moves on in new centrifuge tube.
The applicant once selected four specimen, the extracting method of the two of which specimen present invention to carry out fecal bacteria genomic DNA
Extracting, other two selects traditional method, and result is as shown in the table:
As seen from table, the concentration that the present invention extracts feces genomic DNA is higher, and purity is preferable, and extracts speed.
Embodiment 3
The step of the extracting method of the faecal viruses RNA of the present embodiment is:
1. feces pre-treatment: weigh human or animal feces 200mg in centrifuge tube clean for 2mL, add 500ul～1mL buffer
A, fully broken after, take supernatant 200ul～5ul in new centrifuge tube, in case subsequent experimental.
2. the cracking of microorganism in feces: add 300～500ul buffer B and 20ul buffer in above-mentioned standby centrifuge tube
C, vortex oscillation mix homogeneously, heating in water bath 5～30 minutes under the conditions of 37 DEG C～95 DEG C.
The absorption of 3.RNA: add the buffer E of 20～50ul buffer D and 300～500mL, vortex after having cracked
Vibration fully mixing, stands 5min.Centrifuge tube is put into Magneto separate on magnetic frame, removes the liquid in centrifuge tube.
4. the removal of residual impurity: be sequentially added into 100ul～1000ul buffer F and buffer G, vortex oscillation 3～
30 seconds, fully carry out Magneto separate after mixing, inhale and abandon waste liquid.
The eluting of 5.RNA: addition 30～300ul buffer H in above-mentioned centrifuge tube, vortex oscillation 3～30 seconds, the most mixed
Even, 30～75 DEG C of water-baths 1～10 minutes.Vortex oscillation 3～30 seconds, fully mix, be cooled to room temperature, Magneto separate, will wash
De-liquid moves on in new centrifuge tube.
Reagent used above and consumptive material are the material polluted without RNase, prevent the degraded of RNA in operating process.
Claims (6)
1. paramagnetic particle method fecal bacteria genomic DNA/viral RNA rapid extracting method, it is characterised in that: this extracting method
Step be:
S1, feces pre-treatment: weigh human or animal's feces in centrifuge tube, add buffer A, treat that feces fully crushes laggard
Row stands, until centrifuge tube solid precipitation, have supernatant to separate out time, take supernatant in new centrifuge tube, in case subsequent experimental；
Described buffer A by concentration be 0.1～1M potassium dihydrogen phosphate and sodium chloride solution 1:1 by volume that concentration is 0.1～1M～
5 mix；
The cracking of microorganism in S2, feces: successively add buffer B and buffer C in the centrifuge tube hold primary supernatant,
Vortex oscillation mix homogeneously, heating in water bath under conditions of less than 100° centigrade；Described buffer B is by concentration 0.1～10mol/L
Guanidinesalt, concentration 1～50mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride and the sodium lauryl sulphate of concentration 10～100mmol/L
1:1～3:1～5 mixes by volume；PH value range is 4.0～7.0；Described buffer C is 1～50mg/mL egg
White enzyme K；
The absorption of S3, DNA/RNA: after having cracked, successively adds buffer D and buffer E, and vortex oscillation, fully
Stand after mixing；Then the centrifuge tube after standing is put into and is carried out Magneto separate on magnetic frame, removes the liquid in centrifuge tube, stays
Solids；Described buffer D is magnetic bead；Described buffer E is volumetric concentration 20～the alcoholic solution of 80%；
S4, the removal of residual impurity: successively adding buffer F and buffer G to the centrifuge tube leaving solids, vortex shakes
Swing, fully carry out Magneto separate after mixing；Described buffer F is by concentration 0.1～10mol/L saline solution and concentration 1～50mmol/L
Tri(Hydroxymethyl) Amino Methane Hydrochloride 1:1 by volume～5 mixes, and pH value is 5～10；Described Buffer G be 1～
The Tri(Hydroxymethyl) Amino Methane Hydrochloride solution of 50mmol/L；
The eluting of S5, DNA/RNA: add buffer H, vortex oscillation in the centrifuge tube after processing through step 4, wait to fill
Heating in water bath is carried out after dividing mixing；Then it is vortexed again for vibration, fully mixes, carry out Magneto separate after being cooled to room temperature, contained
There is the eluent of genome NDA/RNA, eluent is moved on in new centrifuge tube standby；Described buffer H be concentration 0.1～
10mol/L HCI solution, pH value is 6～10.
Paramagnetic particle method fecal bacteria genomic DNA/viral RNA rapid extracting method the most according to claim 1, its feature
It is: in step 4, when the pH value range of buffer F is not between 5～10, adds the alcoholic solution of 30%～80%
The pH value range of regulation buffer F is 5～10.
Paramagnetic particle method fecal bacteria genomic DNA/viral RNA rapid extracting method the most according to claim 1, its feature
Being: in step 2, the temperature of heating in water bath is 37 DEG C～95 DEG C；The time of heating in water bath is 10 minutes to 1 hour.
Paramagnetic particle method fecal bacteria genomic DNA/viral RNA rapid extracting method the most according to claim 1, its feature
Being: in steps of 5, the temperature of heating in water bath is 30 DEG C～75 DEG C；The time of heating in water bath is 1 minute to 10 minutes.
Paramagnetic particle method fecal bacteria genomic DNA/viral RNA rapid extracting method the most according to claim 1, its feature
It is: in step 2, in 3,4,5, the time of vortex oscillation is 3～30 seconds.
Paramagnetic particle method fecal bacteria genomic DNA/viral RNA rapid extracting method the most according to claim 1, its feature
Being: in step 3, the time of standing is 1～10min.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967712A (en) * | 2017-05-12 | 2017-07-21 | 广州和实生物技术有限公司 | Faeces DNA rapid extraction kit |
| CN107460190A (en) * | 2017-09-06 | 2017-12-12 | 成都晟博源生物工程有限公司 | A kind of extracting method of bacteria RNA |
| CN108034652A (en) * | 2017-12-18 | 2018-05-15 | 浙江省农业科学院 | The extracting method of microorganism total DNA in a kind of animal and bird intestines |
| CN109266642A (en) * | 2017-07-18 | 2019-01-25 | 天根生化科技（北京）有限公司 | The kit and extracting method of paramagnetic particle method extraction fecal microorganism genome |
| CN112011593A (en) * | 2019-05-30 | 2020-12-01 | 苏州海狸生物医学工程有限公司 | Fecal nucleic acid extraction kit |
| CN114231526A (en) * | 2022-02-23 | 2022-03-25 | 南京瑞贝西生物科技有限公司 | Method for extracting genome DNA of high-abundance fecal microorganisms |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106967712A (en) * | 2017-05-12 | 2017-07-21 | 广州和实生物技术有限公司 | Faeces DNA rapid extraction kit |
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| CN108034652A (en) * | 2017-12-18 | 2018-05-15 | 浙江省农业科学院 | The extracting method of microorganism total DNA in a kind of animal and bird intestines |
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| CN114231526A (en) * | 2022-02-23 | 2022-03-25 | 南京瑞贝西生物科技有限公司 | Method for extracting genome DNA of high-abundance fecal microorganisms |
| CN114231526B (en) * | 2022-02-23 | 2022-08-16 | 南京瑞贝西生物科技有限公司 | Method for extracting genome DNA of high-abundance fecal microorganisms |
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